Han Roelofsen

Biological effects of propionic acid in humans; metabolism, potential applications and underlying mechanisms

Undigested food is fermented in the colon by the microbiota and gives rise to various microbial metabolites. Short-chain fatty acids (SCFA), including acetic, propionic and butyric acid, are the principal metabolites produced. However, most of the literature focuses on butyrate and to a lesser extent on acetate; consequently, potential effects of propionic acid (PA) on physiology and pathology have long been underestimated. It has been demonstrated that PA lowers fatty acids content in liver and plasma, reduces food intake, exerts immunosuppressive actions and probably improves tissue insulin sensitivity. Thus increased production of PA by the microbiota might be considered beneficial in the context of prevention of obesity and diabetes type 2. The molecular mechanisms by which PA may exert this plethora of physiological effects are slowly being elucidated and include intestinal cyclooxygenase enzyme, the G-protein coupled receptors 41 and 43 and activation of the peroxisome proliferator-activated receptor γ, in turn inhibiting the sentinel transcription factor NF-κB and thus increasing the threshold for inflammatory responses in general. Taken together, PA emerges as a major mediator in the link between nutrition, gut microbiota and physiology.

In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance

ABSTRACT Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.

Regulation of adipokine production in human adipose tissue by propionic acid

Eur J Clin Invest 2010; 40 (5): 401–407

Decreased bilirubin transport in the perfused liver of endotoxemic rats.

BACKGROUND/AIMS Hyperbilirubinemia associated with sepsis is frequently observed in humans. In this study, an experimental rat model was developed to study bilirubin metabolism and transport during endotoxemia. METHODS Rats were injected intravenously with a single bolus of lipopolysaccharide (1 mg/kg); after 18 hours, the liver was removed for single-pass perfusion. Unconjugated bilirubin, bilirubin ditaurate (125 nmol/min), and/or taurocholate (1.5 mumol/min) were infused. Rate constants for uptake were determined from the disappearance of a bolus of bilirubin ditaurate in a recirculating perfusion. RESULTS In endotoxemic livers, biliary excretion of bilirubin-glucuronides was reduced by 49% (2.04 +/- 0.2 and 3.99 +/- 0.24 nmol.min-1.g liver-1). Similar results were obtained with bilirubin ditaurate, indicating that the reduced transport is not caused by a reduced conjugation capacity. The rate constant of sinusoidal uptake was significantly reduced during endotoxemia (0.191 +/- 0.034 vs. 0.090 +/- 0.035, respectively). Secretion of taurocholate into bile was also reduced (92 +/- 22 vs. 127 +/- 10 nmol.min-1.g liver-1). CONCLUSIONS In endotoxemic rats, biliary clearance of bilirubin and taurocholate is substantially decreased, suggesting that decreased output of bilirubin-glucuronides is not caused by impaired conjugation but by a reduction in transport.

Localization of the Wilson's disease protein in human liver.

BACKGROUND & AIMS Wilsons disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilsons disease protein in hepatic copper handling, its localization in human liver was investigated. METHODS By use of a specific antibody, localization of the Wilsons disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. RESULTS The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. CONCLUSIONS These results show that in human liver the Wilsons disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion.

Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months. Albumin- and fibrinogen-secreting cells were selected and cloned by limiting dilution to obtain homologous cell populations. The established IHH (immortalized human hepatocyte) cell lines were evaluated for their usefulness in studying the regulation of cell growth and of certain differentiated hepatocyte functions.IHH cells retain several differentiated features of normal hepatocytes. They display albumin secretion at a level comparable to cultured primary human hepatocytes (30 µg albumin/ml per day). A portion of the IHH cells are polarized, forming bile canaliculi-like vacuoles where exogeneous organic anions accumulate. The multidrug resistance (MDR) P-glycoprotein, known to be localized at the canalicular membrane, is also present in these vacuoles. The polarized features allowed the use of IHH cells for the study of localization of the newly characterized multidrug resistance protein MRP1. The homologues of MRP were found in hepatocytes, MRP1 and MRP2 (cMOAT), both functioning in ATP-dependent excretion of anionic conjugates. In differentiated hepatocytes, MRP1 expression is extremely low. In contrast, MRP1 is highly expressed in proliferating IHH cells, where it is localized in lateral membranes. A highly differentiated feature of short-term cultured primary hepatocytes which is not detectable in IHH cells is active uptake of the bile salt taurocholate. Furthermore, IHH cells secrete triglyceride (TG)-rich lipoproteins, apolipoprotein B (0.6 µg/ml per day), and apolipoprotein A-I (1 µg/ml per day). However, they secrete apoB-containing TG-rich lipoproteins mainly in the LDL density range, while short-term cultured primary hepatocytes mainly secrete TG-rich lipoproteins in the VLDL density range.In conclusion, functions that are rapidly lost in short-term hepatocyte cultures are, in general, not displayed by IHH cells. Immortalized human hepatocytes provide a valuable tool for studying the regulation of hepatocyte proliferation-related phenomena.

Propionic acid affects immune status and metabolism in adipose tissue from overweight subjects

Eur J Clin Invest 2012; 42 (4): 357–364

Short-Chain Fatty Acids Stimulate Angiopoietin-Like 4 Synthesis in Human Colon Adenocarcinoma Cells by Activating Peroxisome Proliferator-Activated Receptor γ

ABSTRACT Angiopoietin-like protein 4 (ANGPTL4/FIAF) has been proposed as a circulating mediator between the gut microbiota and fat storage. Here, we show that transcription and secretion of ANGPTL4 in human T84 and HT29 colon adenocarcinoma cells is highly induced by physiological concentrations of short-chain fatty acids (SCFA). SCFA induce ANGPTL4 by activating the nuclear receptor peroxisome proliferator activated receptor γ (PPARγ), as demonstrated using PPARγ antagonist, PPARγ knockdown, and transactivation assays, which show activation of PPARγ but not PPARα and PPARδ by SCFA. At concentrations required for PPARγ activation and ANGPTL4 induction in colon adenocarcinoma cells, SCFA do not stimulate PPARγ in mouse 3T3-L1 and human SGBS adipocytes, suggesting that SCFA act as selective PPARγ modulators (SPPARM), which is supported by coactivator peptide recruitment assay and structural modeling. Consistent with the notion that fermentation leads to PPAR activation in vivo, feeding mice a diet rich in inulin induced PPAR target genes and pathways in the colon. We conclude that (i) SCFA potently stimulate ANGPTL4 synthesis in human colon adenocarcinoma cells and (ii) SCFA transactivate and bind to PPARγ. Our data point to activation of PPARs as a novel mechanism of gene regulation by SCFA in the colon, in addition to other mechanisms of action of SCFA.

Sample Stability and Protein Composition of Saliva: Implications for Its Use as a Diagnostic Fluid

Saliva is an easy accessible plasma ultra-filtrate. Therefore, saliva can be an attractive alternative to blood for measurement of diagnostic protein markers. Our aim was to determine stability and protein composition of saliva. Protein stability at room temperature was examined by incubating fresh whole saliva with and without inhibitors of proteases and bacterial metabolism followed by Surface Enhanced Laser Desorption/Ionization (SELDI) analyses. Protein composition was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation of saliva proteins followed by digestion of excised bands and identification by liquid chromatography tandem mass spectrometry (LC-MS/MS). Results show that rapid protein degradation occurs within 30 minutes after sample collection. Degradation starts already during collection. Protease inhibitors partly prevented degradation while inhibition of bacterial metabolism did not affect degradation. Three stable degradation products of 2937 Da, 3370 Da and 4132 Da were discovered which can be used as markers to monitor sample quality. Saliva proteome analyses revealed 218 proteins of which 84 can also be found in blood plasma. Based on a comparison with seven other proteomics studies on whole saliva we identified 83 new saliva proteins. We conclude that saliva is a promising diagnostic fluid when precautions are taken towards protein breakdown.

Redistribution of canalicular organic anion transport activity in isolated and cultured rat hepatocytes

The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)‐dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly defined by its deficiency in mutant TR− rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione‐bimane (GS‐B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37°C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl‐glutathione (GS‐DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20°C. Residual cMOAT activity in 24‐hour cultured hepatocytes could be further diminished by incubation of the cells with 1 μmol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37°C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport.